DNA extraction

1. School Of Studies in Zoology,
Jiwaji Universality, Gwalior
PROJECT FILE
Topic- Bisulphite Modification and
Methylation Specific PCR
Submitted to, Submitted by,
Head of the Department, Shashank Garg,
Schoolof Studies in Zoology, M.Sc 2nd
Semester
JiwajiUniversity

2. Acknowledgement-
I would like to express my thanks of gratitude to the Head of the department,
ProfessorP.K. Tiwari Sir, who gave me the oppurtunity to work on this project
and guided me throught the process.
I would also like to expand my gratutide to Ms. Anjali Mam and Ms. Nivedita
Mam for their constant guidance and supervision during the whole procedure.
I would also like to express my gratitude to my teachers Dr Jaya Kaushik Mam,
Dr. Sarika Singh Mam, Dr Hemant Samadhiya Sir, Mrs, Neha Sharma Mam,
Mr. Ram kumar Lodhi Sir for the supportand guidance.
Shashank Garg

3. Contents-
1. Introduction
2. Objectives
3. Materials and Methods
4. Observations and Results
5. Discussion
6. Conclusions
7. References

4. Introduction-
Genetics is the branch of biology which deals with the study of genes, genetic variation and
heredity in organisms. The genes present encodes various proteins responsible for various
biological activities in an organism. The genotype i.e. the genetic makeup of an organism is
concerned with the phenotype i.e. external appearance of an organism.
Epigenetics is the study of heritable changes in gene expression that do not involve changes
to the underlying DNA sequence — a change in phenotype without a change in genotype —
which in turn affects how cells read the genes. First introduced by C.H. Waddington in 1942
to name “the causal interactions between genes and their products, which bring the phenotype
into being,” (Esteller 2008). By contrast, Arthur Riggs and colleagues defined epigenetics as
“the study of mitotically and/or meiotically heritable changes in gene function that cannot be
explained by changes in DNA sequence”, in other words, inheritance (Bird 2007). Many
types of epigenetic processes have been identified—they include methylation, acetylation,
phosphorylation, ubiquitylation, and sumolyation (Weinhold 2006).
The best-known epigenetic marker is DNA methylation. Methylation involves attaching
small molecules called methyl groups, each consisting of one carbon atom and three
hydrogen atoms, to segments of DNA. When methyl groups are added to a particular gene,
that gene is turned off or silenced, and no protein is produced from that gene. DNA
methylation has critical roles in the control of gene activity and the architecture of the
nucleus of the cell. In eukaryotes, DNA methylation occurs in cytosines that precede
guanines; these are called dinucleotide CpGs. CpG sites are not randomly distributed in the
genome; instead, there are CpG-rich regions known as CpG islands, which span the 5′ end of
the regulatory region of many genes. These islands are usually not methylated in normal
cells. The methylation of particular subgroups of promoter CpG islands can, however, be
detected in normal tissues (Esteller 2008).
DNA extraction involves separating the nucleic acids in a cell away from proteins and other
cellular materials. Humans, animals, and plants are composed of eukaryotic cells; these cells
also have a lipid bilayer outer membrane and cytoplasm containing proteins, sugars, lipids,
and inorganic ions of various types and function. However, eukaryotic cells also contain
other membrane-enclosed compartments called organelles. Because of the lipid structure of
the cell (and nuclear) membrane(s), presence of proteases and magnesium, and coiling of
DNA around histones, many of the available DNA extraction procedures have common
elements. Indeed, the extraction of DNA generally follows three basic steps; (1) Lyse (break
open) the cells; (2) Separate the DNA from the other cell components and (3) isolation of the
DNA (Elkins 2013).
Quantification of nucleic acids is commonly used to determine the concentration of DNA
which is isolated from various source (either human blood, tissue etc.). DNA exhibits strong
absorbance of UV due to the presence of conjugated double bonds of the constant purine and

5. pyramidine bases and these have characteristics of OD (optical density)of absorbance
maximum at 260-280nm which is linearly related with the concentration of the DNA in the
solution up to the OD value of 1.8-2.0 . The spectroscopic method is used to check the purity
of DNA. Qualitative analysis of the DNA is done by the process of Agarose gel
electrophoresis. Gel electrophoresis separates DNA fragments by size in a solid support
medium (agarose gel). DNA samples are pipetted into the sample wells. Application of
an electric current at the top (anodal, negative) end causes the negatively-charged DNA to
migrate (electrophorese) towards the bottom (cathodal, positive) end. The rate of migration is
proportional to size: smaller fragments move more quickly, and wind up at the bottom of the
gel. DNA is visualized by including in the gel an intercalating dye, ethidium
bromide. DNA fragments take up the dye as they migrate through the gel. Illumination
with ultraviolet light causes the intercalated dye to fluoresce.
Figure- Representation of the chromatin structure, including histones and DNA, which
become available to epigenetic marks.
Source- Epigenetics: Fundamentals, History, and Examples. (n.d.). Retrieved from
https://www.whatisepigenetics.com/fundamentals/
Epigenetic research uses powerful techniques for the study of DNA methylation, such as
sodium bisulfite modification associated with polymerase-chain-reaction procedures.
Bisulfite is known to deaminate cytosine in nucleic acids, while 5-methylcytosine resists this
bisulfite action. For this reason, bisulfite treatment has been used for detecting 5-
methylcytosine in DNA, a minor component of eukaryotic DNA, presently recognized as
playing an important role in the control of gene function. . The deamination of cytosine by
sodium bisulphite and subsequent PCR involves five critical steps: (1) denaturation of the
DNA into single strands;(2) reaction of bisulphite with the 5–6 double bond of cytosine to
give a cytosine sulphonate derivative; (3) hydrolytic deamination of the resulting cytosine–
bisulphite derivative to give a uracil sulphonate derivative; (4) removal of the sulphonate
group by a subsequent alkali treatment to give uracil; and (5) PCR amplification. Because the
conversion of cytosines to uracils creates noncomplementary strands (i.e., uracils opposite

6. guanines), DNA must be amplified with separate pairs of primers that are specific for either
the top or bottom DNA strands. Following PCR amplification, the uracils are amplified as
thymines, whereas 5-MeC residues are amplified as cytosines (Clark, Statham, Strizaker,
Molloy, Frommer 2006).
Figure- Major Steps involved in bisulphite modification, PCR amplification and analysis.
Source-Patterson, Molloy, Qu, Clark 2011.
Methylation specific polymerase chain reaction (MSP) is based on the use of two distinct
methylation specific primer sets for the sequence of interest. The unmethylated (U) primer
will only amplify sodium bisulfite converted DNA in unmethylated condition, while the
methylated (M) primer is specific for sodium bisulfite converted methylated DNA. MSP
provides a positive, sensitive, quick and cost-effective test to analyze the methylation status
of CpG dinucleotides in a CpG-islands making the technique suitable for high-throughput
analysis of clinical samples (Derks, Lentjes, Hellebrekers, Bruïne, Herman, Engeland 2004).
The annealing temperature for specific determination of methylation patterns is critical.
MS relies on the specific annealing of primers based on matches or mismatches of the
primer sequence to bisulfite treated DNA.

7. Objectives-
1. To isolate the DNA from the human blood sample.
2. Quantitative and Qualitative analysis of the isolated
DNA.
3. To do Bisulphite Modification of the isolated DNA and
Methylation specific polymerase chain reaction.

8. Materials and Methodologies-
DNA Extraction-
Requirements-
a. Glass and plastic wares- Micropipettes (P-20, P-200, P-1000), pipette tips,
microfuge tubes,
b. Instruments- Centrifuge, Rotary mixer.
c. Chemicals-
 Extraction buffer (Tris HCL pH 8.0 100 mM, 0.5 EDTA pH 8.0 50 mM,
NaCL 150 mM)
 Proteinase K (20 mg/ml)
 Distilled and buffered saturated phenol
 Chloroform: isoamyl alcohol (24:1)
 3M Sodium acetate (pH 5.2)
 Ethanol (absolute and 70%)
 TE buffer (pH 8.0)
d. Human whole blood
Procedure-
 Take 1 ml of venous blood and 1 ml of lysis buffer.
 Incubate it overnight at 37°C.
 Add equal amount of Tris- saturated phenol.
 Mix well slowly on a rotary mixer and centrifuge at 10,000 rpm for 10 min at room
temperature (RT) to separate the vicious aqueous phase.
 Again add the Tris- saturated phenol, mix well and centrifuge as done in previous
step.
 To the aqueous phase add freshly prepared chloroform: isoamyl alcohol (24:1). Mix
well and centrifuge at 10,000 rpm for 10 min at room temperature to separate the
vicious aqueous phase.
 Transfer the aqueous phase to a new tube, add 3M Sodium acetate (pH 5.3) one-tenth
part of the supernatant and add 2.5 times of chilled ethanol to precipitate DNA. Leave
at 20°C foe 1-2 hours or longer.
 Centrifuge at 10,000 rpm for 10 min at RT. Pour off the supernatant and wash the
pellet with 70% ethanol. Spin at 10,000 rpm for 5 min at 4°C, pour off ethanol, air dry
the pellet and dissolve in about 50µl TE buffer (pH 8.0).
Quantitative Analysis-
 The quantitative analysis is done using a NanoDrop Spectrometer. The drop (2 µl) of
extracted DNA sample is loaded in the instrument and the readings are recorded. All
the readings are measured at 280 nm.

9. Qualitative Analysis by Gel Electrophoresis-
Requirements-
a. Electrophoresis buffer (0.5X Tris Borate EDTA)
b. EtBr (Ethidium bromide 10mg/ml)
c. Agarose (0.8%)
d. Loading buffer (6X)
e. Horizontal gel Electrophoretic Unit
f. Gel casting tray and gel comb
Procedure-
 Seal the edges of clean, dry glass plates with the help of tape to form a mould.
 Prepare sufficient 1X electrophoresis buffer to fill the electrophoresis tank and to
prepare the gel.
 Add the 0.8g of powdered agarose in a flask containing buffer.
 Heat the slurry in a microwave oven until the agarose dissolves.
 Cool the solution to 50-60°C and add EtBr to a final concentration of 0.5µg/ml and
mix thoroughly.
 Position the comb 0.5-1mm above the plate so that a complete well is formed when
the agarose is added.
 Pour the reminder of warm agarose solution into the mould. The gel should be in
between 3-5mm thick. Allow it to polymerise for 15-30 min.
 After the polymerisation remove the tape and carefully remove the comb.
 Add just enough electrophoresis buffer to cover the gel to a depth of 1mm.
 Mix the samples with 2-3µl of loading buffer and load the samples into the wells
using a pipette.
 Attach the electrodes with tank so the DNA will migrate towards the anode.
 After running the sample to an approximate distance, turn off the current and examine
under the UV.
Bi-sulphite Modification-
Requirements-
a. Gold standard kit
b. CT conversion buffer- The CT conversion reagent is supplied as a solid mixture. Add
900µl water, 300µl M dilution buffer, and 50µl dissolving buffer to a tube of CT
conversion reagent. Mix at RT with frequent vortexing or shaking for 10 min.
c. M Wash buffer- Add 24 ml of 100% ethanol to the 6 ml M wash buffer concentrate.
Procedure-

10.  Add 150 µl CT reagent to 20 µl of DNA sample in a PCR tube. Mix the sample by
flicking the tube or pipetting the sample up and down, then centrifuge the liquid to the
bottom of the tube.
 Move the tube in a thermal cycle and perform the following steps-
a. 98°C for 10 minutes
b. 64°C for 2.5 hours
c. 4°C storage upto 20 hours
 Add 600 µl of M binding buffer to a zymo spin column and place the column into a
provided collecting tube.
 Load the sample (from step 2) into the column containing the M binding buffer, close
the cap and mix by inverting the column several times.
 Centrifuge at full speed (10000 rpm) for 30 sec, discard the flow through.
 Add 100 µl of M wash buffer to the column, centrifuge at full speed for 30 seconds.
 Add 200 µl of M desulphonation buffer to the column and let it to stand at RT for 15-
20 minutes. After the incubation centrifuge at full speed for 30 seconds.
 Add 200 µl of M wash buffer to the column, centrifuge at full speed for 30 seconds.
 Add another 200 µl of M wash buffer to the column and centrifuge for additional 30
seconds
 Place the column into a 1.5ml centrifuge tube. Add 10 µl elution buffer directly to the
column matrix. Centrifuge for 30 seconds at full speed to elute the DNA.
Methylation specific PCR-
Requirements-
a. PCR buffer
b. Forward and Reverse primer
c. Template
d. Taq Polymerase
e. NFW
Procedure-
 Add the following reagents in a eppendrof tube-
NFW 13.8 µl
Buffer (5X) 4 µl
Forward Primer 0.5 µl
Reverse Primer 0.5 µl
Template 1 µl
Taq 0.2 µl

11. Observations and Results-
1. The quantitative analysis of the DNA extracted from the human whole blood shows
the following absorbance and A260/A280 ratio at a wavelength of 280 nm-
Sample Absorbance A260/A280 ratio
Sample 1 320 ng/µl 1.8
Sample 2 263 ng/µl 2.1
Sample 3 140 ng/µl 1.84
2. By the Qualitative analysis of the DNA extracted from the human whole blood
following bands are seen on the agarose gel, by the gel electrophoresis, under the UV
light-
3. After the bisulphite modification and subsequent methylation specific polymerase
chain reaction, following bands are seen when the PCR product is loaded on an 2%
agarose gel followed by electrophoresis-
1 2 3 4 5 6 7 8

12. 1. 2. 3.

13. Discussion-
Epigenetic information is defined as heritable information other than the DNA sequence, and
is represented by methylation of cytosines at CpG sites. Modifications in the nucleic acid that
do not change the DNA sequence can affect gene activity. Chemical compounds, added to
single genes can regulate the gene activity; these modifications are termed as epigenetic
changes. The term epigenome is used to define all of the chemical compounds that have been
added to the entirety of one’s DNA or genome as a way to regulate the activity or expression
of all the genes within the genome. The chemical compounds of the epigenome are not part of
the DNA sequence, but are on or attached to DNA. Epigenetic modifications remain as cells
divide and in some cases can be inherited through the generations. Epigenetic changes can help
to determine whether genes are turned on or off and can influence the production of proteins
in certain cells, ensuring that only necessary proteins are produced Aberrant DNA methylation
can be applied to cancer diagnostics in three ways. First, if aberrant methylation of some CGIs
is specifically present in cancer cells, it can be used to detect cancer cells in biopsy samples or
cancer-derived free DNA in plasma. Secondly, if aberrant methylation of some CGIs is
associated with a disease phenotype, such as prognosis, responses to chemotherapies or
occurrence of adverse effects, it can be used as a marker to predict the phenotype. Thirdly, if
aberrant methylation of some CGIs in non-cancerous tissue is associated with a risk for cancer
development. Many examples have been reported for this type of use. Methylation of p16, O6-
methylguanine DNA methyltransferase (MGMT), retinoic acid receptor beta (RARβ), death-
associated protein kinase 1 (DAPK), hMLH1, E-cadherin, APC and RASSF1A was analyzed in
sputum and BAL to detect lung cancers. Methylation of glutathione S-transferase P1 (GSTP1)
was analyzed in urine and ejaculate to detect prostate cancers etc. (Miyamoto, Ushijima 2005)

14. Conclusion-
The heritable change in the gene expression that do not involve change in the underlying DNA
sequence is termed as epigenetics. Various epigenetic processes occurs, methylation being the
prominent one. Some of these methylation can be tumor specific. The bisulphite modification
technique followed by the subsequent methylation specific polymerase chain reaction can be
used to check the methylated and unmethylated regions. The hypermethylated and the
hypomethlayed gene expression can also be assayed using the technique.

15. References-
 Esteller, M. (2008). Epigenetics in cancer. New England Journal of
Medicine, 358(11), 1148-1159.
 Bird, A. (2007). Perceptions of epigenetics. Nature, 447(7143), 396.
 Epigenetics: Fundamentals, History, and Examples. (n.d.). Retrieved from
https://www.whatisepigenetics.com/fundamentals/
 Patterson, K., Molloy, L., Qu, W., Clark, S. DNA Methylation: Bisulphite
Modification and Analysis. J. Vis. Exp.(56), e3170, doi:10.3791/3170 (2011).
 Clark, S. J., Statham, A., Stirzaker, C., Molloy, P. L., & Frommer, M. (2006). DNA
methylation: bisulphite modification and analysis. Nature protocols, 1(5), 2353.
 Derks, S., Lentjes, M. H., Hellebrekers, D. M., De Bruïne, A. P., Herman, J. G., &
Van Engeland, M. (2004). Methylation-specific PCR unraveled. Analytical Cellular
Pathology, 26(5-6), 291-299.
 Elkins, K. M. (2012). Forensic DNA biology: a laboratory manual. Academic Press.
 “QUALITATIVE AND QUANTITATIVE ANALYSIS OF DNA BY
SPECTROPHOTOMETRY.” PharmaTutor,
www.pharmatutor.org/articles/qualitative-and-quantitative-analysis-of-dna-by-
spectrophotometry.
 Miyamoto, K., & Ushijima, T. (2005). Diagnostic and therapeutic applications of
epigenetics. Japanese journal of clinical oncology, 35(6), 293-301.